NISTIR 8143

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report of 

Participant Results: Summer 2015

Comparability Study

(Exercise 11)

Mary Bedner 

This publication is available free of charge from:

http://dx.doi.org/10.6028/NIST.IR.8143
NISTIR 8143

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report of 

Participant Results: Summer 2015

Comparability Study

(Exercise 11)

Mary Bedner

Chemical Sciences Division

Material Measurement Laboratory

This publication is available free of charge from:

http://dx.doi.org/10.6028/NIST.IR.8143

September 2016 

U.S. Department of Commerce 

Penny Pritzker, Secretary

National Institute of Standards and Technology 

Willie E. May, Under Secretary of Commerce for Standards and Technology and Director
i

ABSTRACT

The National Institute of Standards and Technology (NIST) has established a Vitamin D 

Metabolites Quality Assurance Program (VitDQAP) in collaboration with the National

Institutes of Health (NIH) Office of Dietary Supplements. Participants in the eleventh exercise

of this program, the Summer 2015 Comparability Study, were asked to use the methodology of

their choice to measure concentrations of 25-hydroxyvitamin D in pooled human serum control 

and study materials distributed by NIST. The study materials consisted of VitDQAP-I and 

VitDQAP-II (materials designed for the VitDQAP). Standard Reference Material (SRM) 968d

Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum Level 1 was provided as a 

control material. Participants provided their data to NIST, where it was compiled and evaluated

for trueness relative to the NIST value and concordance within the participant community. A 

report of results was provided to all participants of the study, and laboratories were identified by

code numbers known only to them. The results from this eleventh study are reported along with 

a summary of the analytical methods used.

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OVERVIEW OF THE SUMMER 2015 COMPARABILITY STUDY

For the Summer 2015 comparability study of the collaborative National Institute of Standards and 

Technology and National Institutes of Health (NIST/NIH) Vitamin D Metabolites Quality 

Assurance Program (VitDQAP), human serum control and study materials were distributed to 

participants for evaluation. Standard Reference Material (SRM) 968d Fat-Soluble Vitamins, 

Carotenoids, and Cholesterol in Human Serum Level 1 (SRM 968d L1) was provided as a control 

material for assay validation. For SRM 968d L1 (Control), the participants were provided the NIST 

target values within the data reporting sheet so that they could qualify their methods prior to 

analyzing the study samples. The study materials consisted of two vials, each containing a sample 

of pooled human serum. In this study, Vial A was VitDQAP-I, and Vial B was VitDQAP-II, both 

of which contain endogenous levels of the vitamin D metabolites. Participants were asked to 

determine 25-hydroxyvitamin D in each of the human serum control and study samples. Individual 

concentration values for 25-hydroxyvitamin D3 (25(OH)D3), 25-hydroxyvitamin D2 (25(OH)D2), 

and 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3) were requested along with a total concentration 

of 25-hydroxyvitamin D: 25(OH)DTotal = 25(OH)D2 + 25(OH)D3. 

There were 48 participants and 55 datasets (7 participants provided data from two methods) in the 

Summer 2015 comparability study. Seventeen (17) of the datasets originated from immunoassay 

(IA) techniques, including 11 from chemiluminescence immunoassay (CLIA), two from enzyme 

immunoassay (EIA), three from radioimmunoassay (RIA), and one from chemiluminescence

enzyme immunoassay (CLEIA). Appendix A-1 summarizes the IA methods used by the 

participants. Thirty-eight (38) of the datasets originated from liquid chromatographic (LC) 

methods; of those, 33 were from LC with tandem mass spectrometric detection (LC-MS/MS), one 

was from LC-MS, and four were from LC with ultraviolet absorbance detection (LC-UV). The LCMS/MS and LC-MS methods are collectively referred to as LC-MSn

. A summary of the LC 

methods used by the participants may be found in Appendices A-2 and A-3. Note: The 

methodological information provided on the data reporting sheet was used to update the list from 

previous comparability studies. For prior participants that did not provide method details for the 

Summer 2015 study, the information in the appendices were not edited and may not be current.

The raw data received from all participants are summarized in Appendix B. The IA methods do 

not distinguish between 25(OH)D3 and 25(OH)D2, and hence IA participants reported single values 

for 25(OH)DTotal in the control and study materials. The LC methods measure the vitamin D 

metabolites separately, and the majority of the LC participants reported values for 25(OH)D3 in 

addition to 25(OH)DTotal. Thirteen LC participants reported non-zero results for 25(OH)D2 in at 

least one of the study materials, and 11 participants reported results for 3-epi-25(OH)D3. One 

participant also reported values for 24(R), 25-dihydroxyvitamin D3 (24(R),25(OH)2D3), which is 

not represented in Appendix B.

Appendix B also provides the summarized NIST results for each of the serum materials. A detailed

description of the NIST method is provided in the next section of this report.

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SUMMARY OF THE NIST METHOD USED TO EVALUATE THE CONTROL AND 

STUDY MATERIALS

NIST used isotope dilution LC-MS/MS (ID-LC-MS/MS)[1] to determine the vitamin D metabolites 

(25(OH)D3, 25(OH)D2, and 3-epi-25(OH)D3) in the control and study materials evaluated in this 

comparability study. The ID-LC-MS/MS approach is a reference measurement procedure (RMP) 

for 25(OH)D3 and 25(OH)D2 that is recognized by the Joint Committee for Traceability in 

Laboratory Medicine (JCTLM). 

The NIST values for 25(OH)D3, 25(OH)D2, and 3-epi-25(OH)D3 in VitDQAP-I (Vial A) and 

VitDQAP-II (Vial B) are reported with expanded uncertainties (U) that incorporate components for 

measurement variability and measurement uncertainty associated with the density of the materials

and the purity of the reference standards. In addition, the measurements include an additional 1 % 

type B uncertainty for unknown systematic errors, which is consistent with the practice used at 

NIST for clinical measurements [1]. For SRM 968d L1 (Control), the NIST values for 25(OH)D3

and 3-epi-25(OH)D3 are reported as described for VitDQAP-I (Vial A) and VitDQAP-II (Vial B), 

but the value for 25(OH)D2 was well below the limit of quantitation and was estimated to be 

0.1 ng/mL based on one measurement. 

The values for 25(OH)DTotal in VitDQAP-I (Vial A), VitDQAP-II (Vial B) and SRM 968d L1 

(Control) are the sum of the individual values for 25(OH)D3 and 25(OH)D2, and the expanded 

uncertainty incorporates measurement uncertainties for the two analytes.

 

1 Tai, S. S.-C.; Bedner, M.; Phinney, K.W.; Anal. Chem. 2010 82, 1942-1948.

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SUMMER 2015 COMPARABILITY STUDY RESULTS AND DISCUSSION

Results for 25(OH)DTotal

A summary of the individual participant data for total 25-hydroxyvitamin D (25(OH)DTotal) in 

VitDQAP-I (Vial A), VitDQAP-II (Vial B), and SRM 968d L1 (Control) is provided in Table 1.

The community results are summarized at the bottom of Table 1 for all reported methods, the IA 

methods only, the LC methods only, and the LC-MSn methods only. The community results 

include the total number of quantitative values reported (N); the median value; the median absolute 

deviation from the median (MADe), a robust estimate of the standard deviation; and the percent 

coefficient of variation (CV %). 

Table 1 also presents the NIST results for 25(OH)DTotal in the control and the two study materials.

 

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Table 1. Summary of participant and NIST results for 25(OH)DTotal (ng/mL) in VitDQAP-I (Vial 

A), VitDQAP-II (Vial B), and SRM 968d L1 (Control). 

VitDQAP-I VitDQAP-II SRM 968d L1

Lab Method Vial A Vial B Control

026 LC-MS/MS 31.9 38.6 12.4

030a RIA 26.2 37.2 15.8

056a LC-MS/MS 30.7 36.1 12.1

056b LC-MS/MS 32.3 37.4 13.6

060 LC-MS/MS 31.5 35.8 13.7

110 LC-UV 19.5 31.3 12.6

116 LC-MS/MS 35.3 42.1 13.8

150 LC-MS/MS 26.8 32.6 10.2

180 RIA 27.7 32.1 13.9

187 LC-MS/MS 29.9 37.6 12.1

188 CLIA 31.1 36.8 11.9

189 LC-UV 33.5 38.4 11.0

194 LC-MS/MS 31.0 42.0 n/r

196 CLIA 32.1 38.6 14.4

197 LC-MS/MS 33.6 41.1 12.2

198a LC-MS/MS 35.5 45.3 12.7

198c CLIA 32.0 39.2 7.3

199 LC-MS/MS 30.9 38.4 12.6

204b LC-MS/MS 33.6 39.1 12.2

209 LC-MS/MS 31.9 39.4 12.5

211 LC-MS/MS 32.7 39.0 11.3

212 LC-MS/MS 32.8 40.5 13.2

214b CLIA 23.4 31.1 21.1

214c LC-MS/MS 32.6 39.0 12.5

215 LC-MS/MS 30.8 36.0 12.0

216 LC-MS/MS 34.2 43.3 12.8

217 LC-MS/MS 28.4 38.4 15.7

218a CLIA 30.2 45.7 13.5

218b LC-MS/MS 29.2 42.6 12.3

220a LC-MS/MS 34.0 41.5 12.6

221b LC-UV 29.2 34.5 9.6

221c LC-MS 28.4 36.1 11.5

225 LC-MS/MS 34.3 38.0 13.4

228a LC-MS/MS 31.5 39.9 12.5

231b CLIA 36.0 46.0 14.5

243a LC-UV 34.4 34.4 12.2

243b LC-MS/MS 34.5 34.5 12.2

244 LC-MS/MS 29.3 37.4 12.7

249 LC-MS/MS 32.8 39.0 12.8

251 LC-MS/MS 36.0 46.0 n/r

253 LC-MS/MS 37.1 44.0 14.3

255 LC-MS/MS 37.7 47.5 16.4

256 CLIA 27.0 30.9 16.0

258 CLIA 40.4 48.5 18.1

259 LC-MS/MS 30.2 33.0 14.0

261 CLIA 41.5 50.5 22.2

262 CLIA 29.0 38.4 17.7

267 CLEIA 29.9 36.7 12.4

268a RIA 28.2 34.2 13.9

268b EIA 46.7 58.7 28.4

270 LC-MS/MS 29.8 35.5 12.4

271 LC-MS/MS 23.2 36.3 13.0

272 LC-MS/MS 31.5 40.0 12.3

273 EIA 24.2 40.3 14.1

274 CLIA 31.5 48.4 18.5

n/r = not reported or not determined

VitDQAP-I VitDQAP-II SRM 968d L1

Vial A Vial B Control

N 55 55 53

Median 31.5 38.6 12.7

MADe 3.4 3.9 1.2

CV% 11 10 9.3

N 17 17 17

Median 30.2 38.6 14.5

MADe 3.7 9.7 3.1

CV% 12 25 21

N 38 38 36

Median 31.9 38.5 12.5

MADe 3.0 3.6 0.6

CV% 9.5 9.4 4.4

N 34 34 32

Median 31.9 39.0 12.6

MADe 2.7 3.9 0.6

CV% 8.6 9.9 5.0

NIST Value 32.0 37.5 12.5

U 0.8 0.9 0.4

All 

methods

IA 

methods 

LC 

methods LC-MS

n

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For all participant datasets, the single reported values for 25(OH)DTotal in VitDQAP-I (Vial A), 

VitDQAP-II (Vial B), and SRM 968d L1 (Control) are plotted in Figure 1, Figure 2, and

Figure 3, respectively. The results from immunoassay methods are displayed with open dark blue 

circles (○), and the results from the LC-based methods are displayed with open light blue circles 

(○). The results from the individual methods were sorted separately, as indicated by the x-axis 

labels.

From the single reported values for all datasets for a given technique (IA or LC), the consensus 

median and the consensus expanded uncertainty (2  MADe) were determined. For both of the 

major techniques (IA or LC) in each figure, the solid lines () and () represent the 

consensus median, and the dashed lines (- - - - -) and (- - - - -) represent the consensus expanded 

uncertainty interval (median ± 2  MADe). The laboratories with results that fall between the two 

dashed lines are within the consensus range for their technique (IA or LC).

The red lines () in each figure (Figures 1 – 3) represent the NIST value and its associated 

uncertainty (i.e., value ± U). NIST has confidence that the “true” value for each material lies within 

this interval. When these lines are not within the consensus ranges for each technique (IA or LC), 

then there may be method bias. 

Specific results for each of the three study materials are summarized below. Note that the 

assessment is based on the actual reported values, not the lines and symbols, which have been 

enlarged to show detail and the laboratory number.

VitDQAP-I (Vial A): Figure 1

 For the IA results, three reported values are outside of the consensus range (two CLIA, one 

EIA).

 For the LC results, two reported values are outside of the consensus range (one LC-MSn, one

LC-UV). 

 The consensus median value for the IA results is lower than the NIST expanded uncertainty 

range (red lines).

 The consensus median value for the LC results is comparable to the NIST expanded uncertainty 

range (red lines).

 The NIST expanded uncertainty range (red lines) falls within the consensus range for both IA 

and LC. 

VitDQAP-II (Vial B): Figure 2

 For the IA results, the data appear to be non-normally distributed, and the consensus variability 

is not well-described by the MADe estimation; however, one EIA result is outside the consensus

range.

 For the LC results, two LC-MSnvalues are outside the consensus range (both LC-MSn). 

 The consensus median values for both the IA and the LC results are comparable with the NIST 

expanded uncertainty range (red lines).

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SRM 968d L1 (Control): Figure 3

 For the IA results, four reported values are outside of the consensus range (three CLIA, one 

EIA).

 For the LC results, eight reported values are outside of the consensus range (six LC-MSn, two

LC-UV). 

 The consensus median value for the IA results is higher than the NIST expanded uncertainty 

range (red lines).

 The consensus median value for the LC results is comparable to the NIST expanded uncertainty 

range (red lines).

 The NIST expanded uncertainty range (red lines) falls within the consensus range for both IA 

and LC. 

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Figure 1. Participant and NIST results for 25(OH)DTotal in VitDQAP-I (Vial A) as determined by immunoassay (CLIA, EIA, RIA, 

and CLEIA) and LC (LC-MSnand LC-UV) methods. 

 IA method laboratory values 

 IA method consensus range encloses ± 2 MADe around consensus median

 LC method laboratory values

 LC method consensus range encloses ± 2 MADe around consensus median

 NIST value range encloses expanded uncertainty interval 

271

150

217221c

218b244270187259056a215199194060272228a026209056b214c211212249

197204b220a216225243b

116198a251

253255

110

221b

189

243a

214b

256

262

218a

188274198c196

231b

258

261

273

268b

030a

180268a

267

10

20

30

40

50

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330

25(OH)DTotal in VitDQAP-I "Vial A" (ng/mL)

CLIA EIA RIA CLEIA LC-M Sn LC-UV

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Figure 2. Participant and NIST results for 25(OH)DTotal in VitDQAP-II (Vial B) as determined by immunoassay (CLIA, EIA, RIA, 

and CLEIA) and LC (LC-MSnand LC-UV) methods. 

150259

243b

270060215056a221c271

244056b187225199217026211214c249204b209228a272212197220a194116218b216253

198a251

255

110

243a221b

189

256214b

188

262196198c

218a231b

274258

261

273

268b

180

268a

030a267

10

20

30

40

50

60

70

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330

25(OH)DTotal in VitDQAP-II "Vial B" (ng/mL)

CLIA EIA RIA CLEIA LC-M Sn LC-UV

 IA method laboratory values 

 IA method consensus range encloses ± 2 MADe around consensus median

 LC method laboratory values

 LC method consensus range encloses ± 2 MADe around consensus median

 NIST value range encloses expanded uncertainty interval 

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Figure 3. Participant and NIST results for 25(OH)DTotal in SRM 968d Level 1 (Control) as determined by immunoassay (CLIA, EIA,

RIA, and CLEIA) and LC (LC-MSnand LC-UV) methods. 

 IA method laboratory values 

 IA method consensus range encloses ± 2 MADe around consensus median

 LC method laboratory values

 LC method consensus range encloses ± 2 MADe around consensus median

 NIST value range encloses expanded uncertainty interval 

150

211221c

215056a187243b197204b218b272026270209214c228a199220a198a244216249271212225056b060116259253

217

255

221b

189

243a

110

214b

256

262

218a188

274

198c

196231b

258

261

273

268b

030a180

268a

267

5

10

15

20

25

30

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330

25(OH)DTotal in SRM 968d L1 "Control" (ng/mL)

CLIA EIA RIA CLEIA LC-M Sn LC-UV

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Figure 4 presents direct graphical comparisons of the 25(OH)DTotal results for a) VitDQAP-I (Vial 

A) and VitDQAP-II (Vial B), and b) VitDQAP-II (Vial B) and SRM 968d L1 (Control). In each 

plot, there are two blue consensus boxes, one for IA methods and one for LC methods (as 

indicated). Laboratory results that are within the consensus range for both study materials are 

within the blue consensus boxes. Conversely, laboratory results that fall outside of (or on the edge 

of) either of the consensus boxes are not included in the consensus ranges and are highlighted with 

their laboratory code numbers. In each plot, the NIST values for the materials are denoted with a 

red diamond symbol (), and the Youden line (y=x) centered on the NIST value is illustrated by a 

red line () across the magnitude of the y-axis and x-axis, respectively.

Specific results as assessed from the Youden comparison plots are summarized below.

VitDQAP-I (Vial A) and VitDQAP-II (Vial B): Figure 4 a

 IA results that are not included in the consensus ranges include: 258, 261, and 268b.

 LC results that are not included in the consensus ranges include: 110, 251, 255 and 271.

 The Youden line runs through the center of the LC consensus box and near the center of the IA 

consensus box, illustrating that both the IA and LC results are in agreement with each other and 

with the NIST results for these materials.

 The linear trend (results closely aligned with the Youden line) indicates participant-specific 

analytical bias.

VitDQAP-II (Vial B) and SRM 968d L1 (Control): Figure 4 b

 The consensus box for the IA results is extremely large for these two materials, which hinders 

an assessment of the outliers; however, the IA results that are not included in the consensus 

ranges include numbers 198c, 214b, 261, and 268b. 

 LC results that are not included in the consensus ranges include numbers 116, 150, 189, 217, 

221b, 253, 255, and 259.

 The Youden line runs through the center of the LC consensus box and through the bottom of the 

IA consensus box, illustrating that the LC results are in better agreement with the NIST results 

than are the IA results for these materials.

 The lack of strong linear trend suggests either significant differences between SRM 968d L1 

(Control) and VitDQAP-II (Vial B) (e.g., concentration difference) or the ‘attractor’ effect of 

participants knowing the correct value for the control. 

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Figure 4. Youden comparison 

plot of the results for 

25(OH)DTotal in a) VitDQAP-I 

(Vial A) and VitDQAP-II (Vial 

B) and b) VitDQAP-II (Vial B) 

and SRM 968d L1 (Control) 

for all methods. 

116

150

189

198c

211

214b

217

221b

253

255

259

261

5

10

15

20

25

10 20 30 40 50 60

25(OH)DTotal in SRM 968d L1 "Control" (ng/mL)

25(OH)DTotal in VitDQAP-II "Vial B" (ng/mL)

268b

(28.4)

LC methods

IA methods

110

251

255

258

261

268b

271

10

20

30

40

50

60

10 20 30 40 50

25(OH)DTotal in VitDQAP-II "Vial B" (ng/mL)

25(OH)DTotal in VitDQAP-I "Vial A" (ng/mL)

IA methods

LC methods

a

b

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Discussion of Results for 25(OH)DTotal

In the Summer 2015 comparability study, both study materials VitDQAP-I (Vial A) and VitDQAPII (Vial B) and SRM 968d L1 (Control) contain predominantly 25(OH)D3 as the metabolite 

contributing to 25(OH)DTotal. The CV %’s of 11 %, 10 %, and 9.3 % (all methods) for VitDQAP-I 

(Vial A), VitDQAP-II (Vial B), and (Control), respectively, are consistent with participant 

performance for other materials containing predominantly 25(OH)D3 that were evaluated in 

previous comparability studies of the VitDQAP.

The Summer 2015 exercise was the second to utilize study materials that were evaluated in previous 

comparability studies of the VitDQAP. VitDQAP-I (Vial A) was also evaluated in the Summer 

2014 comparability study (Vial A), and VitDQAP-II (Vial B) was previously evaluated in Summer 

2013 (Vial A). Table 2 provides the program results for each of these two study materials for the 

labs participating in the current study. Using the results in Table 2, the participant performance for 

these materials over time can be assessed. When the summary statistics at the bottom of Table 2

are compared, the median and CV % results are generally consistent across both comparability 

studies in which the materials were evaluated with the exception of the IA results for VitDQAP-II, 

which has a significantly higher CV % of 25 % in the current study, compared to 6.3 % in Summer 

2013. The higher CV % in the present study is attributable to the non-normal distribution of the IA 

results, which led to an overestimation of the MADe and the resulting CV % (see Figure 2). 

Figure 5 presents direct graphical comparisons of the 25(OH)DTotal results for 25(OH)DTotal in a) 

VitDQAP-I (Vial A) in the present study (Summer 2015) and in a previous study (Summer 2014) 

and b) VitDQAP-II (Vial B) in the present study (Summer 2015) and in a previous study (Summer 

2013). The features of the plots are the same as described for Figure 4. The clustering of results 

around the NIST value in both Figure 5 a and b illustrates that there are not consistent withinlaboratory biases for VitDQAP-I and VitDQAP-II over 1 and 2 years, respectively, and that the 

within-round variability is consistent with the over-time variability. While the vast majority of labs 

yield results that are within the consensus boxes for their techniques, the labs that fall outside are 

not in as good statistical control. For VitDQAP-I, these labs include 110, 188, 258, and 261 

(Figure 5a), and for VitDQAP-II, these labs include 030a, 110, 188, 198a and 255 (Figure 5b).

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Table 2. Summary of participant data for 25(OH)DTotal (ng/mL) in VitDQAP-I in the current study 

(Vial A) and a prior study (Summer 2014) as well as for VitDQAP-II in the current study (Vial B) 

and a prior study (Summer 2013).

Summer 2015 Summer 2013

Vial B Vial A

38.6 X

37.2 33.6

36.1 36.4

37.4 X

35.8 39.4

31.3 30.1

42.1 36.7

32.6 X

32.1 X

37.6 39.6

36.8 47.0

38.4 X

42.0 43.4

38.6 40.9

41.1 33.9

45.3 49.7

39.2 40.8

38.4 41.5

39.1 X

39.4 42.4

39.0 42.0

40.5 X

31.1 39.6

39.0 36.1

36.0 40.4

43.3 38.2

38.4 37.2

45.7 37.5

42.6 42.3

41.5 39.0

34.5 X

36.1 X

38.0 44.6

39.9 34.6

46.0 X

34.4 X

34.5 X

37.4 36.5

39.0 36.4

46.0 X

44.0 41.7

47.5 50.1

30.9 X

48.5 X

33.0 X

50.5 X

38.4 X

36.7 X

34.2 X

58.7 X

35.5 X

36.3 X

40.0 X

40.3 X

48.4 X

VitDQAP-II

Summer 2015 Summer 2013

Vial B Vial A

55 29

38.6 39.6

3.9 4.2

10 10

17 6

38.6 40.2

9.7 2.5

25 6.3

38 23

38.5 39.4

3.6 4.3

9.4 11

34 22

39.0 39.5

3.9 4.2

9.9 11

37.5 37.5

0.9 0.9

VitDQAP-II

Summer 2015 Summer 2013

Vial A Vial A

N 55 44

Median 31.5 32.7

MADe 3.4 3.4

CV% 11 10

N 17 11

Median 30.2 30.4

MADe 3.7 3.9

CV% 12 13

N 38 33

Median 31.9 33.4

MADe 3.0 3.9

CV% 9.5 12

N 34 29

Median 31.9 33.4

MADe 2.7 3.9

CV% 8.6 12

NIST Value 32.0 32.0

U 0.8 0.8

VitDQAP-I

IA 

methods 

LC 

methods LC-MS

n All 

methods

Summer 2015 Summer 2014

Lab Method Vial A Vial A

026 LC-MS/MS 31.9 30.7

030a RIA 26.2 35.1

056a LC-MS/MS 30.7 33.4

056b LC-MS/MS 32.3 30.3

060 LC-MS/MS 31.5 28.0

110 LC-UV 19.5 32.5

116 LC-MS/MS 35.3 35.1

150 LC-MS/MS 26.8 28.2

180 RIA 27.7 30.4

187 LC-MS/MS 29.9 33.8

188 CLIA 31.1 42.9

189 LC-UV 33.5 39.4

194 LC-MS/MS 31.0 33.9

196 CLIA 32.1 29.8

197 LC-MS/MS 33.6 30.3

198a LC-MS/MS 35.5 X

198c CLIA 32.0 X

199 LC-MS/MS 30.9 31.4

204b LC-MS/MS 33.6 30.8

209 LC-MS/MS 31.9 34.0

211 LC-MS/MS 32.7 37.8

212 LC-MS/MS 32.8 31.9

214b CLIA 23.4 29.4

214c LC-MS/MS 32.6 31.3

215 LC-MS/MS 30.8 34.8

216 LC-MS/MS 34.2 37.2

217 LC-MS/MS 28.4 39.2

218a CLIA 30.2 31.1

218b LC-MS/MS 29.2 38.1

220a LC-MS/MS 34.0 31.0

221b LC-UV 29.2 34.3

221c LC-MS 28.4 X

225 LC-MS/MS 34.3 32.9

228a LC-MS/MS 31.5 37.0

231b CLIA 36.0 X

243a LC-UV 34.4 29.8

243b LC-MS/MS 34.5 30.0

244 LC-MS/MS 29.3 34.0

249 LC-MS/MS 32.8 30.4

251 LC-MS/MS 36.0 37.0

253 LC-MS/MS 37.1 30.8

255 LC-MS/MS 37.7 33.4

256 CLIA 27.0 33.0

258 CLIA 40.4 33.9

259 LC-MS/MS 30.2 34.4

261 CLIA 41.5 24.2

262 CLIA 29.0 27.5

267 CLEIA 29.9 30.4

268a RIA 28.2 X

268b EIA 46.7 X

270 LC-MS/MS 29.8 X

271 LC-MS/MS 23.2 X

272 LC-MS/MS 31.5 X

273 EIA 24.2 X

274 CLIA 31.5 X

X= did not participate in that study

VitDQAP-I

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14

Figure 5. Youden comparison 

plot of the results for 25(OH)DTotal

(ng/mL) in a) VitDQAP-I (Vial A) 

in the present study (Summer 

2015) and in a previous study 

(Summer 2014 – Vial A) and b) 

VitDQAP-II (Vial B) in the present 

study (Summer 2015) and in a 

previous study (Summer 2013 –

Vial A). 

030a

110

188

198a255

15

25

35

45

55

15 25 35 45 55

25(OH)DTotal in VitDQAP-II "Vial A" Summer 2013

25(OH)DTotal in VitDQAP-II "Vial B" Summer 2015

IA methods

LC methods

110

188

258

261

10

20

30

40

50

10 20 30 40 50

25(OH)DTotal in VitDQAP-I "Vial A" Summer 2014

25(OH)DTotal in VitDQAP-I "Vial A" Summer 2015

IA methods

LC methods

a

b

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15

LC Results for 3-Epi-25(OH)D3

Of the two major techniques IA and LC, only the LC methods can independently measure the 

individual metabolites 25(OH)D2, 25(OH)D3 and 3-epi-25(OH)D3. In the Summer 2015 

comparability study of the VitDQAP, the study materials and the control contained negligible 

amounts of the 25(OH)D2 metabolite, and hence the 25(OH)DTotal values reported in Table 1

generally reflect the 25(OH)D3 concentrations measured by the LC participants. However, both the 

VitDQAP-I (Vial A) and VitDQAP-II (Vial B) study materials contained measurable concentrations 

of the 3-epi-25(OH)D3 metabolite, which does not contribute to the reported 25(OH)DTotal values.

Of the 38 LC participants in the Summer 2015 comparability study, 11 reported values for 3-epi25(OH)D3 in at least one of the materials. The study results and the NIST values for 3-epi25(OH)D3 are presented in Table 3. For each material, the median LC result agrees well with the 

NIST value.

Table 3. Summary of participant and NIST results for 3-epi-25(OH)D3 in VitDQAP-I (Vial A), 

VitDQAP-II (Vial B), and SRM 968d L1 (Control). 

VitDQAP-I VitDQAP-II SRM 968d L1

Lab Method Vial A Vial B Control

026 LC-MS/MS 1.9 3.6 0.7

056a LC-MS/MS 1.6 2.8 0.5

060 LC-MS/MS 1.8 3.2 0.7

150 LC-MS/MS 1.7 3.2 0.5

204b LC-MS/MS n/d 3.5 n/d

216 LC-MS/MS 1.4 3.0 0.7

228a LC-MS/MS 1.8 2.7 0.7

243a LC-UV 1.7 1.9 n/d

243b LC-MS/MS 1.6 2.0 n/d

249 LC-MS/MS 1.6 3.2 0.8

272 LC-MS/MS 1.8 3.5 0.8

N 10 11 8

Median 1.7 3.2 0.67

MADe 0.1 0.5 0.12

CV% 7 17 19

N 9 10 8

Median 1.7 3.2 0.67

MADe 0.1 0.5 0.12

CV% 7 16 19

NIST Value 1.7 3.2 0.65

U 0.1 0.1 0.03

LC 

methods LC-MS

n

n/d = not detected

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16

Dihydroxyvitamin D3 Metabolite

This is the second comparability study in which a participant reported results for at least one of the 

dihdroxy metabolites, 24(R),25(OH)2D3, in each of the study materials. The results provided by 

participant 60 for this metabolite include:

24(R),25(OH)2D3

(ng/mL)

VitDQAP-I (Vial A) 2.44

VitDQAP-II (Vial B) 6.82

SRM 968d L1 (Control) 1.02

NIST has developed a candidate RMP for the determination of 24(R),25(OH)2D3 and has assigned 

reference values for this metabolite in SRM 972a. However, NIST is not providing values for 

24(R),25(OH)2D3 for the VitDQAP study materials at this time. 

Conclusions from the Summer 2015 Comparability Study of the VitDQAP

The Summer 2015 comparability study was the 11th exercise for the VitDQAP. Over 11 studies, the 

participant performance has been consistent for study materials that contain predominantly 

25(OH)D3; the CV has been in the range from 7 % to 19 %, and the median values (all methods) 

have been biased slightly high or were comparable to the NIST values. In the Summer 2015 

comparability study, VitDQAP-I (Vial A), VitDQAP-II (Vial B), and SRM 968d L1 (Control) also 

contain predominantly 25(OH)D3 and follow these longstanding trends. In addition, Summer 2015 

represents the second study in which both VitDQAP-I (Vial A) and VitDQAP-II (Vial B) were 

evaluated in the VitDQAP. Table 2 and Figure 5 contains the program results for this material in 

both studies and demonstrates the consistency of the participant results for these study materials.

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17

Appendix A-1. Summary of immunoassay methods as reported by the study participants.

Laboratory 

Number IA Method Sample Preparation Vendor/kit*

30a RIA Samples were extracted with acetonitrile A

180 RIA Samples were extracted with acetonitrile A

188 CLIA n/r B

196 CLIA No sample preparation required C

198c CLIA n/r n/r

214b CLIA n/r C

218a CLIA Direct analysis C

231b CLIA n/r B

256 CLIA n/r C

258 CLIA n/r D

261 CLIA No sample preparation required D

262 CLIA n/r E

267 CLEIA n/r F

268a RIA n/r G

268b EIA n/r H

273 EIA n/r n/r

274 CLIA n/r D

n/r = not reported

*NIST cannot endorse or recommend commercial products, therefore individual vendors/kits are indicated with a unique letter but not identified

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18

Appendix A-2. Summary of LC-MSn methods as reported by the study participants. 

Laboratory 

Number

Internal 

Standard (IS) Sample Preparation Chromatographic Conditions Detection: MRM ions

26

25(OH)D2-d6 and 

25(OH)D3-d6

Liquid-liquid extraction method

PFP column (100 mm × 3.2 mm); 

isocratic elution with 82 % 

methanol/18 % water; 

flow 0.4 mL/min

25(OH)D3 401/365; 

25(OH)D2 413/355;

3-epi-25(OH)D3 401/365

56a

25(OH)D2-d3; 

25(OH)D3-d6; 

3-epi-25(OH)D3-d3

Samples were extracted with 

hexane, evaporated, then 

reconstituted with 69 % methanol

PFP column (100 mm × 2.1 mm; 

1.9 μm); isocratic elution; 

flow 0.4 mL/min

25(OH)D3 383/365; 

25(OH)D3-d6 389/371; 

25(OH)D2 395/377; 

25(OH)D2-d3 398/380; 

3-epi-25(OH)D3 383/365; 

3-epi-25(OH)D3-d3 386/368

56b n/r n/r n/r n/r

60

25(OH)D3 -d6

25(OH)D3-d3

24,25(OH)2D3-d6

IS was added, and then samples 

were extracted with acetonitrile, 

evaporated, and reconstituted 

with 90 % methanol/10 % water

PFP column (100 mm × 3.0 mm; 

2.6 μm); gradient with water, 

methanol and acetonitrile (0.05 % 

formic acid)

25(OH)D3 383/211; 

25(OH)D3-d6 389/211; 

25(OH)D2 413/355; 

3-epi-25(OH)D3 401/383

116 25(OH)D3

 -d6

Serum proteins were precipitated 

with methanol

Online SPE; reversed-phase 

column; isocratic elution with 

95 % methanol/5 % water; 

flow 0.6 mL/min

25(OH)D3 383/211; 

25(OH)D3-d6 389/211; 

25(OH)D2 395/269

150

25(OH)D2-d6 and 

25(OH)D3-d3

Sample (200 μL) was mixed with 

IS solution, liquid-liquid extracted, 

centrifuged, supernatant 

evaporated, and reconstituted in 

mobile phase

PFP column (100 mm × 3.0 mm; 

2.6 μm); isocratic separation with 

74 % methanol/26 % water 

(2 mmol/L ammonium acetate, 

0.1 % formic acid); 

flow 0.5 mL/min

25(OH)D3 401/383, 401/365; 

25(OH)D2 413/395, 413/365

187

deuterated 

standards for 

25(OH)D2 and 

25(OH)D3

SPE

C18 column (50 mm × 2.1 mm; 

3 μm); gradient with methanol and 

water

25(OH)D2 413/395; 

25(OH)D3 401/383

194 25(OH)D3

 -d6

Proteins precipitated with 

acetonitrile, top layer removed, 

evaporated, and reconstituted 

with methanol

C8 column (50 mm × 2 mm); 

isocratic elution with 70 % 

acetonitrile/ 30 % water; 

flow 0.7 mL/min

25(OH)D2 395/119; 

25(OH)D3 383/211

197 25(OH)D3

 -d6

Precipitating agent added (200 μL 

with 20 ng IS) to each serum 

sample (200 μL), calibrator and 

control sample followed by mixing, 

centrifugation, and analysis

C18 column (50 mm × 4.6 mm; 

5 μm); column temp 45 °C; gradient 

with water and methanol; 

flow 1.0 mL/min

n/r

198a 25(OH)D3

 -d6

Proteins precipitated with 

methanol, followed by ZnSO4 

addition, hexane extraction, 

centrifugation, evaporation under 

N2, and reconstitution in methanol 

(0.1 % formic acid)

C18 column (50 mm x 2.1 mm; 

3.5 μm); isocratic elution with 

85 % methanol (0.1 % formic acid); 

flow 0.5 mL/min

25(OH)D3 401/383, 401/365; 

25(OH)D2 413/395, 413/355; 

25(OH)D3-d6 407/389, 

407/371

199 proprietary proprietary proprietary proprietary

204b

25(OH)D2-d3; 

25(OH)D3-d6; 

3-epi-25(OH)D3-d3

Protein crash with 73 % methanol 

followed by liquid-liquid extraction 

with hexane, centrifugation, 

evaporation, and reconstitution in 

mobile phase

PFP column (100 mm × 2.1 mm; 

1.9 μm); column temperature 30 °C; 

isocratic elution with 73 % 

methanol/27 % water; 

flow 0.4 mL/min

APCI

25(OH)D3 383/365, 383/257;

25(OH)D2 395/377, 395/209;

3-epi-25(OH)D3 383/365, 

383/257

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19

209 25(OH)D3

 -d6

Proteins were precipitated with 

5 % ZnSO4 in methanol

C8 column (50 mm × 2 mm; 5 μm); 

gradient with water/methanol; flow 

0.7 mL/min

APCI

25(OH)D3 383/229,383/211; 

25(OH)D3-d6 389/211; 

25(OH)D2 395/269, 395/119

211 25(OH)D3

-d6

Proteins precipitated with 

acetonitrile containing IS followed 

by centrifugation

Turbulent flow column (32 mm x 

4.6 mm; 3 μm)

25(OH)D3 383/365 (quant), 

383/257 (qual); 25(OH)D2 

395/209 (quant), 395/377 

(qual)

212 25(OH)D3

-d6

Serum (100 μL) proteins 

precipitated using 5 % 

methanol/95 % acetonitrile 

containing the IS (350 μL)

C8 column (50 mm × 2 mm; 

3 μm); gradient of 60 % to 98 % 

acetonitrile (0.1 % formic acid)

25(OH)D3 383/229, 383/211; 

25(OH)D2 395/269, 395/119

214c 25(OH)D3

-d6

Samples were extracted with 

hexane, centrifuged, evaporated, 

and filtered

Column (50 mm × 2.1 mm); isocratic 

elution with 85 % methanol/ 15 % 

water/ 0.1 % formic acid; 

flow 0.3 mL/min

25(OH)D3 401/383; 

25(OH)D3-d6 407/389; 

25(OH)D2 413/395

215 25(OH)D3

-d6

Protein precipitation with 

methanol/isopropanol and ZnSO4; 

supernatant extracted using SPE

C18 column (50 mm × 2.1 mm; 

2.6 μm) column; gradient with water 

(0.1 % formic acid, 5 mmol/L 

ammonium formate) and methanol 

(0.05 % formic acid)

ESI

25(OH)D3 401/383; 

25(OH)D2 413/395; 

25(OH)D3-d6 407/389

216

Derivatized 

deuterated 

standard

Samples extracted using liquidliquid extraction then labeled with 

a derivatization reagent

Reversed-phase column (150 mm × 

2.1 mm); gradient from 25 % water 

(0.05 % formic acid) to 50 % 

acetonitrile (0.05 % formic acid); 

flow 0.2 mL/min

n/r

217 25(OH)D3

-d6

Protein precipitation with ZnSO4 in 

methanol followed by SPE

C8 column (50 mm × 2.1 mm; 

1.7 μm); gradient of 70 % to 98 % 

methanol (with 0.1 % formic acid); 

flow 0.4 mL/min

25(OH)D3 401/159 (quant), 

401/383 (qual); 25(OH)D2 

413/83 (quant), 413/395 

(qual)

218b

From vendorsupplied vitamin D 

kit

Samples were extracted, 

separated, centrifuged, and 

evaporated.

C18 column (50 mm × 2.1 mm; 

1.7 μm); isocratic elution with 

methanol and water; 

flow 0.45 mL/min

25(OH)D3 298

220a

25(OH)D2-d3 and 

25(OH)D3-d6

Protein crash with 90 % methanol/ 

10 % ZnSO4 and then acetonitrile/ 

1 % formic acid; sample filtered; 

phospholipids removed with SPE

C18 column (20 mm × 2.1 mm; 

2.7 μm); gradient with water and 

acetonitrile; flow 1 mL/min; column 

40 °C

MRM with dehydrated 

precursor and product ions

221c

25(OH)D2-d6 and 

25(OH)D3-d3

Protein crash with acetonitrile 

containing IS; SPE extraction; 

elution with methanol/acetonitrile 

solution; evaporation; 

reconstitution with acetonitrile

PFP column (50 mm × 3.0 mm; 

2.7 μm); elution with 

methanol/water/formic acid; column 

40 °C

LC-MS SIM

25(OH)D3 383; 

25(OH)D2 395; 

25(OH)D3-d6 389;

25(OH)D2-d6 401

225 25(OH)D3

-d6 Liquid-liquid extraction PFP column (100 mm × 2.1 mm); 

gradient with methanol/water

25(OH)D3 401/107; 

25(OH)D2 413/83

228a n/r n/r n/r n/r

243b 25(OH)D3

-d6

Samples (400 μL) were mixed with 

solution containing the IS (400 μL)

and the mobile phase (500 μL); 

samples were centrifuged; 

supernatant was diluted; portion 

(50 μL) was injected

PFP column (150 mm × 2 mm); 

isocratic separation with 85 % 

methanol/15 % water; 

flow 0.3 mL/min

25(OH)D3 383/257; 

25(OH)D2 395/269; 

25(OH)D3-d6 389/263;

244 25(OH)D3

-d6

Protein precipitation followed by 

filtration

CN column; mobile phase 

consisting of distilled water (formic 

acid) and methanol

25(OH)D3 383/211; 

25(OH)D3-d6 389/211; 

25(OH)D2 395/269

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20

249

25(OH)D2-d3; 

25(OH)D3-d6; 

3-epi-25(OH)D3-d3

Serum was deproteinated with 

NaOH and 90 % acetonitrile/ 10 % 

methanol followed by SPE

PFP column (100 mm × 2.1 mm; 

1.8 μm); gradient separation with 

water (2 mmol/L ammonium 

acetate) and methanol; 

flow 0.35 mL/min

25(OH)D3 401/159; 

25(OH)D2 413/159

251

25(OH)D2-d3 and 

25(OH)D3-d3

Protein precipitation followed by 

SPE

Phenyl column (50 mm × 2.1 mm; 

1.7 μm); gradient with water and 

methanol (0.1 % formic acid, 

2 mmol/L ammonium acetate); flow 

0.45 mL/min

25(OH)D3 401/159 (quant), 

401/365 (qual); 25(OH)D2 

413/83 (quant), 413/355 

(qual); 25(OH)D3-d3 404/162; 

25(OH)D2-d3 416/358

253

25(OH)D2-d3 and 

25(OH)D3-d3

The sample was extracted, 

centrifuged, and derivatized

C18 column (150 mm × 2.1 mm); 

gradient separation with water and 

methanol; flow 0.4 mL/min

25(OH)D2 588; 

25(OH)D3 576

255 deuterium labeled 

compound

Samples were extracted and 

derivatized with 4-phenyl-1,2,4-

triazoline-3,5-dione

Reversed-phase column (50 mm × 

2.1 mm); gradient with methanol; 

flow 0.5 mL/min

25(OH)D3 607/298; 

25(OH)D2 619/298

259 25(OH)D3

-d6

Liquid-liquid extraction using 

hexane

C8 column; gradient with 

methanol/water/0.1 % formate; 

column temperature 40 °C

25(OH)D3 401/355; 

25(OH)D2 413/355; 

25(OH)D3-d6 407/371

270 25(OH)D3

-d6

Samples were precipitated, 

centrifuged, evaporated, 

reconstituted, centrifuged, and 

upper layer injected

C18 column (300 mm × 4.6 mm; 

3.5 μM); isocratic separation with 

50 % water/ 50 % methanol; 

flow 1.0 mL/min

25(OH)D3 401/383; 

25(OH)D2 413/395; 

25(OH)D3-d6 407/389

271 25(OH)D3

-d6 Protein precipitation

C8 column (3 μm); gradient with 

water/acetonitrile/0.1 % formic acid; 

flow 0.7 mL/min

25(OH)D3 383/229; 

25(OH)D2 395/269

272 Isotopically labeled 

internal standards

Samples were precipitated and 

centrifuged before injection

Analytical column and trap column 

from a kit; separation using a binary 

gradient system and an additional 

isocratic pump

25(OH)D3 383/365, 383/299; 

IS (1): 386/257, 386/232; 

25(OH)D2 395/269, 395/251; 

3-epi-25(OH)D3 383/257, 

383/299;

3-epi-25(OH)D2 395/269, 

395/251; 

IS (2): 386/257, 386/232

C18 = octadecyl; C8 = octyl; PFP = pentafluorophenyl; SPE = solid phase extraction; CN = cyano; 

MRM = multiple reaction monitoring; quant/qual = quantitative/qualitative ions; n/r = not reported;

APPI = atmospheric pressure photoionization; APCI = atmospheric pressure chemical ionization; ESI = electrospray ionization

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21

Appendix A-3. Summary of LC-UV methods as reported by the study participants.

Laboratory 

Number

Internal 

Standard (IS) Sample Preparation Chromatographic Conditions Wavelength

110 n/a

Samples (500 μL) were mixed with 

ethanol (500 μL), extracted twice 

with hexane/methylene chloride 

(5:1), evaporated, and 

reconstituted

C18 column (2.1 mm × 100 mm; 

1.8 μm); gradient with 

acetonitrile/methanol (85:15) and 

isopropanol (100 %)

267 nm

189 unidentified Protein precipitation followed by 

SPE

Reversed-phase column (150 mm × 

4.6 mm); isocratic separation; flow 

0.7 mL/min

265 nm

221b laurophenone

Protein crash with acetonitrile 

solution containing IS, followed by 

SPE, elution with 

methanol/acetonitrile solution, 

evaporation, and reconstitution 

with acetonitrile

CN column (150 mm × 5 mm;

3.5 μm); elution with 

methanol/water/formic acid; column 

temperature 47 °C 

275 nm

243a dodecanophenone

Samples (400 μL) were mixed with 

solution containing the IS 

(400 μL), precipitation reagent 

was added (500 μL), and portion 

of upper layer (50 μL) was 

injected

C18 column (100 mm × 3 mm); 

isocratic elution with water and 

isobutanol; flow 1.2 mL/min; column 

temperature 25 °C

264 nm

C18 = octadecyl; SPE = solid phase extraction; CN = cyano; n/a = not applicable

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22

Appendix B. Raw 

participant data and 

NIST results for 

25(OH)D2, 

25(OH)D3, 

25(OH)DTotal, and

3-epi-25(OH)D3 in 

VitDQAP-I (Vial 

A), VitDQAP-II

(Vial B), and SRM 

968d L1 (Control).

VitDQAP-I VitDQAP-II SRM 968d L1 VitDQAP-I VitDQAP-II SRM 968d L1 VitDQAP-I VitDQAP-II SRM 968d L1 VitDQAP-I VitDQAP-II SRM 968d L1

Lab Method Vial A Vial B Control Vial A Vial B Control Vial A Vial B Control Vial A Vial B Control

026 LC-MS/MS <1.0 <1.0 <1.0 31.9 38.6 12.4 31.9 38.6 12.4 1.9 3.6 0.7

030a RIA n/a n/a n/a n/a n/a n/a 26.2 37.2 15.8 n/a n/a n/a

056a LC-MS/MS 0.6 0.6 0.6 30.1 35.5 11.5 30.7 36.1 12.1 1.6 2.8 0.5

056b LC-MS/MS 0.7 < 0.6 < 0.6 31.7 37.4 13.6 32.3 37.4 13.6 n/r n/r n/r

060 LC-MS/MS 0.7 0.4 0.2 30.8 35.4 13.5 31.5 35.8 13.7 1.8 3.2 0.7

110 LC-UV n/r n/r n/r n/r n/r n/r 19.5 31.3 12.6 n/r n/r n/r

116 LC-MS/MS <3.3 <3.3 <3.3 35.3 42.1 13.8 35.3 42.1 13.8 <4 <4 <4

150 LC-MS/MS 0.7 0.4 0.0 26.1 32.2 10.2 26.8 32.6 10.2 1.7 3.2 0.5

180 RIA n/a n/a n/a n/a n/a n/a 27.7 32.1 13.9 n/a n/a n/a

187 LC-MS/MS <1.5 <1.5 <1.5 29.9 37.6 12.1 29.9 37.6 12.1 n/r n/r n/r

188 CLIA n/a n/a n/a n/a n/a n/a 31.1 36.8 11.9 n/a n/a n/a

189 LC-UV n/r n/r n/r 33.5 38.4 11.0 33.5 38.4 11.0 n/r n/r n/r

194 LC-MS/MS <7 <7 n/r 31.0 42.0 n/r 31.0 42.0 n/r n/r n/r n/r

196 CLIA n/a n/a n/a n/a n/a n/a 32.1 38.6 14.4 n/a n/a n/a

197 LC-MS/MS <5 <5 0.0 33.6 41.1 12.2 33.6 41.1 12.2 n/r n/r n/r

198a LC-MS/MS <5 <5 <5 35.5 45.3 12.7 35.5 45.3 12.7 n/r n/r n/r

198c CLIA n/a n/a n/a n/a n/a n/a 32.0 39.2 7.3 n/a n/a n/a

199 LC-MS/MS <2.0 <2.0 <2.0 30.9 38.4 12.6 30.9 38.4 12.6 n/r n/r n/r

204b LC-MS/MS n/d n/d n/d 33.6 39.1 12.2 33.6 39.1 12.2 n/d 3.5 n/d

209 LC-MS/MS <1.0 <1.0 <1.0 31.9 39.4 12.5 31.9 39.4 12.5 n/r n/r n/r

211 LC-MS/MS 0.0 0.0 0.0 32.7 39.0 11.3 32.7 39.0 11.3 n/r n/r n/r

212 LC-MS/MS <2 <2 <2 32.8 40.5 13.2 32.8 40.5 13.2 n/r n/r n/r

214b CLIA n/a n/a n/a n/a n/a n/a 23.4 31.1 21.1 n/a n/a n/a

214c LC-MS/MS 1.0 0.4 0.2 31.6 38.6 12.3 32.6 39.0 12.5 n/r n/r n/r

215 LC-MS/MS <2 <2 <2 30.8 36.0 12.0 30.8 36.0 12.0 n/r n/r n/r

216 LC-MS/MS 0.8 0.5 0.2 33.4 42.8 12.6 34.2 43.3 12.8 1.4 3.0 0.7

217 LC-MS/MS n/d n/d 1.3 28.4 38.4 14.4 28.4 38.4 15.7 n/r n/r n/r

218a CLIA n/a n/a n/a n/a n/a n/a 30.2 45.7 13.5 n/a n/a n/a

218b LC-MS/MS 0.0 0.0 0.0 29.2 42.6 12.3 29.2 42.6 12.3 n/r n/r n/r

220a LC-MS/MS <5 <5 <5 34.0 41.5 12.6 34.0 41.5 12.6 n/r n/r n/r

221b LC-UV 0.0 0.0 0.0 29.2 34.5 9.6 29.2 34.5 9.6 n/r n/r n/r

221c LC-MS 0.0 0.0 0.0 28.4 36.1 11.5 28.4 36.1 11.5 n/r n/r n/r

225 LC-MS/MS <5 <5 <5 34.3 38.0 13.4 34.3 38.0 13.4 n/r n/r n/r

228a LC-MS/MS n/r n/r n/r 31.5 39.9 12.5 31.5 39.9 12.5 1.8 2.7 0.7

231b CLIA n/a n/a n/a n/a n/a n/a 36.0 46.0 14.5 n/a n/a n/a

243a LC-UV 0.9 0.3 n/d 33.4 34.1 12.2 34.4 34.4 12.2 1.7 1.9 n/d

243b LC-MS/MS 0.9 0.5 n/d 33.6 34.1 12.2 34.5 34.5 12.2 1.6 2.0 n/d

244 LC-MS/MS <5 <5 <5 29.3 37.4 12.7 29.3 37.4 12.7 n/r n/r n/r

249 LC-MS/MS 0.0 0.0 0.0 32.8 39.0 12.8 32.8 39.0 12.8 1.6 3.2 0.8

251 LC-MS/MS <4 <4 n/r 36.0 46.0 n/r 36.0 46.0 n/r n/r n/r n/r

253 LC-MS/MS 0.7 0.5 0.2 36.4 43.5 14.1 37.1 44.0 14.3 n/r n/r n/r

255 LC-MS/MS 0.4 0.2 0.0 37.3 47.3 16.4 37.7 47.5 16.4 n/r n/r n/r

256 CLIA n/a n/a n/a n/a n/a n/a 27.0 30.9 16.0 n/a n/a n/a

258 CLIA n/a n/a n/a n/a n/a n/a 40.4 48.5 18.1 n/a n/a n/a

259 LC-MS/MS n/d n/d <2 30.2 33.0 14.0 30.2 33.0 14.0 n/r n/r n/r

261 CLIA n/a n/a n/a n/a n/a n/a 41.5 50.5 22.2 n/a n/a n/a

262 CLIA n/a n/a n/a n/a n/a n/a 29.0 38.4 17.7 n/a n/a n/a

267 CLEIA n/a n/a n/a n/a n/a n/a 29.9 36.7 12.4 n/a n/a n/a

268a RIA n/a n/a n/a n/a n/a n/a 28.2 34.2 13.9 n/a n/a n/a

268b EIA n/a n/a n/a n/a n/a n/a 46.7 58.7 28.4 n/a n/a n/a

270 LC-MS/MS 2.3 2.3 1.6 27.5 33.2 10.8 29.8 35.5 12.4 n/r n/r n/r

271 LC-MS/MS <4 <4 <4 23.2 36.3 13.0 23.2 36.3 13.0 n/r n/r n/r

272 LC-MS/MS 0.7 0.4 0.0 30.8 39.6 12.3 31.5 40.0 12.3 1.8 3.5 0.8

273 EIA n/a n/a n/a n/a n/a n/a 24.2 40.3 14.1 n/a n/a n/a

274 CLIA n/a n/a n/a n/a n/a n/a 31.5 48.4 18.5 n/a n/a n/a

NIST Value 0.68 0.44 0.1* 31.3 37.1 12.4 32.0 37.5 12.5 1.7 3.2 0.65

U 0.06 0.04 --- 0.8 0.9 0.4 0.8 0.9 0.4 0.1 0.1 0.03

*estimated value (no uncertainty determined)

3-epi-25(OH)D3 (ng/mL)

n/a = not applicable (for immunoassay methods); n/r = not reported or not determined; n/d = not detected; < X = less than a reported quantitation limit of X

25(OH)D3 (ng/mL) 25(OH)DTotal 25(OH)D (ng/mL) 2 (ng/mL)

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This publication is available free of charge from: http://dx.doi.org/10.6028/NIST.IR.8143